Infectious Diseases of Poverty

Infectious Diseases of Poverty
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Human Ebola virus infection in West Africa: a review of available therapeutic
agents that target different steps of the life cycle of Ebola virus
Infectious Diseases of Poverty 2014, 3:43
doi:10.1186/2049-9957-3-43
Kang Yiu Lai ([email protected])
Wing Yiu Ng ([email protected])
Fan Fanny Cheng ([email protected])
ISSN
Article type
2049-9957
Opinion
Submission date
2 September 2014
Acceptance date
13 November 2014
Publication date
28 November 2014
Article URL
http://www.idpjournal.com/content/3/1/43
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Human Ebola virus infection in West Africa: a
review of available therapeutic agents that target
different steps of the life cycle of Ebola virus
Kang Yiu Lai1*
*
Corresponding author
Email: [email protected]
Wing Yiu George Ng1
Email: [email protected]
Fan Fanny Cheng2
Email: [email protected]
1
Department of Intensive Care, Queen Elizabeth Hospital, HKSAR, B6, 30
Gascoigne Rd, Kowloon, Hong Kong SAR, China
2
Department of Medicine, Queen Elizabeth Hospital, HKSAR, Kowloon, Hong
Kong SAR, China
Abstract
The recent outbreak of the human Zaire ebolavirus (EBOV) epidemic is spiraling out of
control in West Africa. Human EBOV hemorrhagic fever has a case fatality rate of up to
90%. The EBOV is classified as a biosafety level 4 pathogen and is considered a category A
agent of bioterrorism by Centers for Disease Control and Prevention, with no approved
therapies and vaccines available for its treatment apart from supportive care. Although
several promising therapeutic agents and vaccines against EBOV are undergoing the Phase I
human trial, the current epidemic might be outpacing the speed at which drugs and vaccines
can be produced. Like all viruses, the EBOV largely relies on host cell factors and
physiological processes for its entry, replication, and egress. We have reviewed currently
available therapeutic agents that have been shown to be effective in suppressing the
proliferation of the EBOV in cell cultures or animal studies. Most of the therapeutic agents in
this review are directed against non-mutable targets of the host, which is independent of viral
mutation. These medications are approved by the Food and Drug Administration (FDA) for
the treatment of other diseases. They are available and stockpileable for immediate use. They
may also have a complementary role to those therapeutic agents under development that are
directed against the mutable targets of the EBOV.
Multilingual abstract
Please see Additional file 1 for translations of the abstract into the six official working
languages of the United Nations.
Background
The recent outbreak of the human Zaire ebolavirus (EBOV) infection starting in West
African countries has resulted in 15,351 infected patients, as of 18st of November 2014. A
total of 5,459 deaths have been reported in six affected countries (Guinea, Liberia, Mali,
Sierra Leone, Spain, and the United States of America) and two previously affected countries
(Nigeria and Senegal) [1]. Apart from supportive care, neither a licensed vaccine nor a
specific therapy is available for the treatment of the human EBOV infection [2]. The World
Health Organization (WHO) has considered that it is ethically acceptable to offer unproven
interventions that have shown promising results in laboratory and animal models, but have
not yet been evaluated for safety and efficacy in humans as potential sources of treatment or
prevention [3]. Several promising therapeutic agents have been identified for the treatment
and immunization of the EBOV. These may include monoclonal antibody (mAbs)-based
therapies (e.g. ZMapp), anti-sense phosphorodiamidate morpholino oligomers (PMO AVI6002), lipid nanoparticle small interfering RNA (LNP-siRNA: TKM-Ebola), and an EBOV
glycoprotein-based vaccine using live-attenuated recombinant vesicular stomatitis virus
(rVSV-EBOGP) or a chimpanzee adenovirus (rChAd-EBOGP)-based vector. Human trial
results of these agents would not be available until next year. Moreover, existing supplies of
all these experimental medications and vaccines for compassionate use are either extremely
limited or exhausted [4-6]. To combat such an unprecedented global public-health crisis
before these experimental agents are available, alternative available interventions that can
target different steps in the replication cycle of the EBOV should be explored in the
management of the human EBOV infection as contingency preparation for the international
dissemination of the EBOV outbreak in West Africa. We have reviewed currently available
therapeutic agents that have shown to be effective in suppressing the proliferation of the
EBOV in cell cultures or animal studies. We propose a therapeutic regimen to supplement the
current supportive therapy aiming to reduce viral load, the most important factor in the
determination of mortality. Through viral load suppression, we may be able to prolong a
patient’s survival in order to provide a better chance for the patient to develop natural
immune defense against the EBOV.
Discussion
The genome of the Ebola virus
The EBOV is an enveloped filamentous RNA virus belonging to the family Filoviridae. The
19-kb linear, non-segmented, negative-sense, single-stranded RNA genome of the EBOV
encodes seven structural proteins and two non-structural proteins in the following order
within the genome: 3′ non-coding region (leader), nucleoprotein (NP), virion protein 35
(VP35), VP40, 3 glycoproteins (sGP/ssGP/GP1,2), VP30, VP24, RNA-dependent RNApolymerase protein (L-polymerase), and 5′ non-coding region [7].
The glycoproteins of the Ebola virus
The EBOV genome encodes one transmembrane protein GP1,2 (GP1–GP2) and two secreted
non-structural proteins: secretary glycoprotein (sGP) and small soluble glycoprotein (ssGP).
A small soluble delta peptide (∆-peptide) is secreted from EBOV-infected cells after the
carboxyl-terminal cleavage of sGP [8]. GP1,2 is produced through transcriptional RNA
editing as a precursor for 676 amino acid polyprotein (GP0), which is post-translationally
cleaved by furin into two disulfide-linked subunits; a surface subunit, GP1; and a membranespanning subunit, GP2. GP1 contains the receptor-binding domain (RBD) for host cell
attachment and a mucin-like domain to protect the RBD from humoral and cell-mediated
immunity. The RBD responsible for receptor binding, viral entry, and cellular tropism is
covered by a heavily glycosylated “glycan cap.” The transmembrane GP2 contains a helical
heptad-repeat region, transmembrane anchor, and a 4-residue cytoplasmic tail. The GP2
drives fusion of the viral membrane with the endosomal membrane of the target cell. This
GP1–GP2 heterodimer then assembles as a trimer on the viral surface. This homotrimeric
GP1,2 complex forms the spike on the envelope membrane of the mature viral particles.
During processing, GP1,2 are unstable, and an abundant amount of a soluble non-virion form
of GP1 and a scanty amount of GP1,2 are released into the circulation [9-12]. The virusassociated GP1,2 and not the other soluble glycoproteins released during the virus infection
are responsible for primary target cell activation [13]. The highly glycosylated mucin-like
region of GP1 is cytotoxic to the host cells [14]. The shedding of souble GP1,2-like protein
due to cleavage of EBOV glycoprotein on the surface of EBOV-infected cells by tumor
necrosis factor-alpha converting enzyme (TACE) can activate non-infected dendritic cells
and macrophages to induce cytokine dysregulation and endothelial cell dysfunction [15]. The
GP2 of the EBOV is able to counter the interferon (IFN)-inducible antiviral protein tetherin
which restricts the VP40-dependent budding of the progeny viral particles from infected cells
[16-18]. The sGP is produced from non-edited mRNA species through furin cleavage from a
precursor pre-sGP. The sGP shares the N-terminal 295 amino acids with GP1, but differs in
the carboxyl terminus by 69 amino acids. The sGP is released into the circulation in the form
of homodimers in anti-parallel orientation [19] to evade an antibody-associated innate
immune response [20,21]. The sGP has an anti-inflammatory function and impairs the
transmigration and activation of neutrophils [22,23]. While the GP1,2 in its particleassociated form mediates endothelial cell activation and a decrease in endothelial cell barrier
function, sGP protects the endothelial cell against cytokine-induced barrier dysfunction. The
sGP constitutes at greater than 80% of the total GP synthesized during infection. Hence, the
hypersecretion of the sGP may protect the EBOV against host humoral immune defense and
the host endothelial cell against cytokine-induced cytotoxicity during the early phase of the
EBOV infection [15,24,25]. ∆-peptide released in EBOV-infected cells joins cathepsins and
integrins to inhibit further entry of the EBOV in a dose-dependent manner to prevent
superinfection of EBOV-infected cells. ∆-peptide inhibits entry of both marburgviruses and
the EBOV, indicating that they might interfere with a common pathway used by filoviruses to
gain entry into target cells [26]. The ssGP of a yet undefined function is produced through
transcriptional editing and secreted in the form of a disulfide-linked homodimer that is
exclusively N-glycosylated. While ssGP appears to share similar structural properties with
sGP, it does not appear to have the same anti-inflammatory function as sGP [22,23,27].
The life cycle of the Ebola virus
The EBOV, being a RNA virus with limited coding capacity, has utilized the host’s unique
metabolic pathway for its viral entry, replication, and egress. The entry of the EBOV into
cells is initiated by interaction of the viral GP1 with host cell surface T-cell immunoglobulin
and mucin domain 1 (TIM-1) receptors. Upon receptor binding, the EBOV is internalized
into endosomes primarily via macropinocytosis [28-30]. Within the acidified endosome
compartment of the host cell, the heavily glycosylated GP1 is cleaved to a smaller 19-kDa
fusogenic form by the low pH-dependent cellular proteases Cathepsin L (CatL) and B (CatB),
exposing residues in the receptor binding site. This allows the binding of GP1 to cholesterol
transporter Niemann-Pick C1 (NPC1), a step in the late endosome phase essential for virus-
host membrane fusion and viral entry [31-34]. Cells where the NPC1 function has been
biochemically disrupted or cells lacking NPC1 showed resistance to the EBOV infection.
Cells from subjects with NPC1 disease were resistant to the EBOV because of defects in the
NPC1 protein [35-38]. After complete fusion of the viral and host endosomal membranes via
conformational change in GP2, viral RNA and its associated proteins are released into the
host cell cytoplasm [39]. Once inside the cytoplasm of the host cell, the EBOV suppresses the
innate immune response via VP35 and VP24 proteins [40], and hijacks transcription and
translation for robust genome replication and the production of new virions. The
ribonucleoprotein (RNP) complex that mediates transcription and replication of the EBOV
genome comprises NP, VP35, VP30, and L protein [41-44]. VP30 is essential in the initiation
of the EBOV transcription, but is not required for viral replication. However, dynamic
phosphorylation of VP30 is an important mechanism to regulate the balance between the
transcription and replication processes in the EBOV replication cycle [45-47]. This unique
property of VP30 allows the development of a genetically stable VP30 deleted EBOV
vaccine with protective efficacy in the mice and guinea pig models [48]. The matrix proteins
VP40 and VP24 associated with the viral lipid coat are important for virus structure and
stability. Both matrix proteins VP24 and VP40 contribute to the regulation of viral genome
replication and transcription [49] and the budding of the virus [50-52], an important step prior
to viral egress [53,54]. This distinct replication cycle of the EBOV serves as an attractive
target for the development of therapeutic agents against the EBOV (see Figure 1 and Table
1).
Figure 1 Schematic diagram showing the replication cycle of Ebola virus (EBOV): Upon
receptor binding of EBOV GP1 with host TIM-1 receptor, EBOV is internalized into
endosomes via macropinocytosis. Within the acidified endosome compartment of the host
cell, under the action of the low pH-dependent cellular proteases cathepsins, the receptor
binding site of GP1 to cholesterol transporter Niemann-Pick C1 (NPC1) is exposed. This
results in conformational change in GP2 , leading to complete fusion of the viral and host
endosomal membranes in the late endosome and the release of viral RNA and its associated
proteins into the host cell cytoplasm. EBOV then hijacks transcription and translation for
robust genome replication and viral protein production under the action of ribonucleoprotein
polymerase complex (RNP polymerase). The accumulation of GP1,2 in the endoplasmic
reticulum leads to endoplasmic reticulum overload response (ER-overload) which, in turn,
induces cytokine dysregulation via the activation of nuclear factor kappa B (NFκB) through
the production of reactive oxygen species (ROS). New virions are released through ATPdependent budding and egress from host cell membrane. Currently available therapeutic
agents that target the different steps of the EBOV life cycle are described in Table 1.
Table 1 Available therapeutic agents that target the different steps of the EBOV life
cycle as shown in the diagram
Medication
Convalescent blood serum
Na+/K+ exchanger
- Amiloride
Chloroquine1
Cationic amphiphiles
Amiodarone1
Dronedarone1
Verapamil2
Clomiphene
Toremifene1
Interferon- beta (IFN-β)
Mechanism of action
Contain neutralizing antibodies to provide passive immunity.
Inhibit virus uptake by macropinocytosis
Leads to alkalinization of endosomes and prevent the acid pH-dependent cleavage
of Ebola virus GP1,2 by endosomal proteases cathepsin B and L.
Induce a Niemann-Pick C-like phenotype and block the entry of EBOV through
late endosomes.
Induce interferon-inducible transmembrane proteins (IFITMP) production to
restrict entry of EBOV.
Suppress viral RNA polymerase.
Inhibit Na+/K+-ATPase that are important in the budding and egress of
encapsulated EBOV.
Favipiravir
Na+/K+/ATPase pump inhibitors
Ouabain
Digoxin
Digitoxin
Anti-oxidants
Suppress ROS-dependent NFκB activation and cytokine dysregulation induced by
GP1,2-induced ER-overload.
High dose N-acetylcysteine infusion
1: Chloroquine, Amiodarone, Dronedarone and Toremifene administration is associated with an increased risk of QT
prolongation and Torsades de pointes. 2: Verapamil should be avoided in patient with hypotension.
Pathogenesis of the Ebola virus infection
Human EBOV hemorrhagic fever, characterized by uncontrolled viral replication together
with immune and vascular dysregulation, has a case fatality rate of up to 90% [7]. Type I
alpha/beta interferons (IFN-α/β), encoded by a single IFN-β and 13 homologous IFN-α genes
in humans, represent an essential element of host defense against virus infections, including
the EBOV [55]. The human EBOV infection is associated with robust IFN-α production—
with plasma concentrations of IFN-α that greatly (60- to 100-fold) exceed those observed in
other viral infections—but limited IFN-β production [56]. The EBOV, protected from the
host interferon response by its encoded VP35 and VP24 proteins [40,57-59], produced a
heavy viral load [60-62], cytopathic damages [14,63,64], and cytokine dysregulation in
humans [65-68]. The efficient productive replication of the EBOV inside monocyte and
macrophages leads to a massive release of proinflammatory cytokines/chemokines and
reactive oxygen species (ROS) [13,15,65,66,69-71], which in turn leads to diffuse endothelial
cell dysfunction [72-76], disseminated intravascular coagulation [77-79], and vasomotor
collapse [80-82]. The infection of the antigen presenting dendritic cells [83-86] and profound
bystander apoptosis of lymphocytes [63,87-89] impairs the development of adaptive
immunity [90,91] and EBOV-specific CD8+ T [92-94], as well as CD4+ T cells [95] that are
important for the clearance of, and protection from, the EBOV infection. Infected monocytederived dendritic cells were impaired in the secretion of pro-inflammatory cytokines, the upregulation of co-stimulatory molecules, and the stimulation of T cells [96]. Numbers of CD4+
and CD8+ T cells are substantially reduced in fatal human and nonhuman primate (NHP)
infections before death [63,88,97].
Immune evasion by the glycoproteins of the Ebola virus: implications on
passive immunization and vaccine development
The EBOV is able to counteract both humoral and cell-mediated immunity through its GP1,2
and sGP [11,98]. The overexpression of mature GP1,2 on the plasma membrane results in the
masking of antigenic epitopes on GP1,2 itself and the shielding of MHC-I and integrin β,
leading to evasion of antiviral immunity. Steric shielding of surface epitopes by the heavily
glycosylated GP impairs the recognition and killing of EBOV-infected cells by the natural
killer and cytotoxic CD8+ T cell during an acute viral infection. It may also contribute to the
persistent infection in the natural reservoir host to perpetuate the spread of the EBOV [99101]. The sGP can evade host antibody-mediated response through “antigenic subversion” by
eliciting non-neutralizing antibodies that cross-react with GP1,2. Thus, the massive secretion
of sGP by the EBOV may prevent effective neutralization of the virus during an EBOV
infection and reduce the effectiveness of vaccines that rely upon neutralizing antibody
responses against GP1,2 [20,21]. Some of the antibodies against GP1 may lead to
enhancement of infectivity of the EBOV via interaction with complement component C1q, a
phenomenon known as the antibody-dependent enhancement. The EBOV initiates infection
by binding its GP1 to its specific human receptor sites on the surface of human cells. The
interaction of C1q enhances binding between the virus-antibody complex and the C1q ligands
on the cell surface, promoting interaction between the EBOV and its receptor. These
infectivity-enhancing antibodies were virus species specific and were primarily correlated
with immunoglobulin IgG2a and IgM levels, but not with IgG1 levels [102,103]. The
presence of infectivity-enhancing antibodies against GP1,2 in the EBOV infection raises
concerns about the effectiveness of GP-based EBOV vaccines, and the use of passive
prophylaxis or treatment with GP-based antibodies [104,105].
Antibodies against GP1 of the EBOV can be neutralizing, enhancing, or non-neutralizing and
non-enhancing. Neutralizing antibodies are produced in infection by the EBOV at a relatively
low frequency [106]. Some anti-EBOV antibodies are known to be neutralizing in vitro but
not protective in vivo, whereas other antibodies are known to be protective in animal models
in vivo, but not neutralizing in vitro [107]. Investigations of anti-GP antibodies against the
EBOV showed that non-neutralizing antibodies recognized GP epitopes in the sGP or nonessential mucin-like domain of GP1, while neutralizing antibodies were specific to RBD in
GP1 or conformation-dependent epitopes at the base of the GP1,2 spike where GP1 meets
GP2. Two neutralizing antibodies (KZ52 and JP3K11) against EBOV—that recognize
conformation-dependent epitopes comprising residues in GP1 and GP2—were identified to
have quite distinct mechanisms of neutralization. KZ52 is a human recombinant IgG1
neutralizing antibody derived from a human survivor of a natural EBOV infection during the
1995 outbreak in Kikwit, Democratic Republic of Congo. KZ52 has impaired recognition for
the sGP and binding was dependent on the presence of GP2 residues which are not present in
the sGP. KZ52 is able to inhibit cathepsin cleavage of GP1,2. JP3K11, a monkey derived
neutralizing monoclonal antibody against EBOV, recognized the cleaved, fusion-active form
of GP [108]. 16 F6 is a mice derived monoclonal IgG1 antibody that neutralizes Sudan
EBOV by preventing the conformational changes in GP1,2 required for membrane fusion.
Both 16 F6 and KZ52 recognize GP1–GP2-bridging epitopes at the base of the GP1,2 trimer,
indicating that this overlapping epitope may be one of the key sites for neutralization of the
EBOV, and is thus a target for immunotherapy and a key goal of vaccine design [109].
Antibody subclass may be another important factor in protection against the EBOV. IgG2
isotype may offer more effective protection against EBOV [110,111]. Although fully
protecting guinea pigs from infection, KZ52 fails to slow viral replication and protect NHPs
from the EBOV infection [112]. In contrast, rVSV-EBOGP [113-116] and rChAd-EBOGP
[117-120]-based vaccination have demonstrated both prophylactic and post-exposure
protection in NHPs [121]. This was previously attributed to the protective action of EBOVspecific CD4+ and CD8+ T-cell response induced by these vaccines in limiting infection and
the inability of KZ52 to completely block all entries of the EBOV into cells and its
subsequent explosive replication [112]. rChAd-EBOGP-based vaccination is able to generate
potent humoral and cell-mediated responses. Significant antibody titers are detectable at 48
weeks post vaccination [122,123]. CD8+ cell-mediated immunity has been shown to play a
critical role in protection against the EBOV infection in NHPs in rChAd-EBOGP-based
vaccination [124]. On the other hand, humoral rather than the cell-mediated response
contributes to protection against the EBOV infection in NHPs in rVSV-EBOGP-based
vaccination [125,126].
Candidate vaccines expressing the EBOV GP or NP protect rodents and NHPs from the lethal
EBOV infection [127-129]. Humoral and cell-mediated immune responses are working
together to provide protection against the lethal EBOV infection. Either response alone may
be able to limit virus replication but both arms of the immune response are required to clear
the infection [97,130]. VP proteins (VP24, VP30, VP35, and VP40) are poor inducers of cellmediated immunity and are inaccessible to the protective effect of VP-induced neutralizing
antibodies because they are not found on the surface of virions or infected cells [131].
However, the genetic sites of these internal proteins are susceptible to siRNA and PMO
interference. TKM-Ebola (a siRNA targeting L-polymerase, VP24, and VP35) can be
administered intravenously or subcutaneously in a lyophilized lipid nanoparticle formulation.
TKM-Ebola offers post-exposure protection against the EBOV infection in NHPs. The FDA
has approved an “expanded access” program for the use of TKM-Ebola in patients with
confirmed or suspected infections [132,133]. Anti-sense phosphorodiamidate morpholino
oligomers AVI-6002 effectively reduce viral load, diminish virally-induced pathology, and
improve survival of NHPs with the EBOV infection by targeting VP24 and VP35 mRNA.
Through judicious placement of positive charges on the drug backbone, the drug is able to
bind to a negative charge on the virus even if binding at one or more drug-virus base pairs are
lost through mutation. This integration of dual targeting and charge complementation
significantly lowers the likelihood of drug resistance through viral mutagenesis [134,135].
Available drugs that target the different steps of the Ebola virus life cycle
Currently available therapeutic agents that are effective in targeting the EBOV infection in
cell or animal studies may include convalescent plasma, favipiravir, chloroquine,
amiodarone, dronedarone, verapamil, clomiphene, toremifene, IFN-β, Na+/K+ exchangers,
Na+/K+-ATPase pump inhibitors, and antioxidants. Except for convalescent plasma and
favipiravir, most of the therapeutic agents under review are acting against the non-mutable
targets of the host cells which participate in the replication cycle of the EBOV. They may
also have a complementary role to conventional therapy in the management of the current
EBOV outbreak in West African countries (see Table 1).
(1) Convalescent blood serum
The WHO issued a consensus statement that the use of whole blood therapies and
convalescent blood serum needs to be considered as a matter of priority in the recent EBOV
outbreak in West African countries [2]. The development of neutralizing antibodies and Tcell responses are important for recovery from the EBOV infection [97,136]. Patients who are
able to mount an immune response to the EBOV will begin to recover in seven to ten days
and start a period of prolonged convalescence [137]. In survivors, early and increasing levels
of IgG, directed mainly against the NP and the VP40, were followed by the clearance of
circulating viral antigen and activation of cytotoxic T cells. In contrast, fatal infection was
characterized by impaired humoral responses, with absent specific IgG and barely detectable
IgM [63]. Convalescent blood has been shown to improve survival of EBOV-infected
patients during the outbreak in Kikwit in 1995 [138]. Immunity against EBOV GP is
sufficient to protect individuals against infection, and several vaccines based on EBOV GP
are under development including recombinant adenovirus, parainfluenza virus, Venezuelan
equine encephalitis virus, vesicular stomatitis virus, and virus-like particles [139].
Neutralizing human monoclonal antibodies is able to protect mouse and guinea pigs from
lethal EBOV. However, the protection was achieved only by treatment shortly before or after
viral infection [140-142]. The EBOV can rapidly mutate to produce antibody-escape mutants.
Hence, antibody therapy may require hyperimmune polyclonal serum or a panel of
monoclonal antibodies of different epitope specificities to be successful [143,144]. These
studies have laid the foundation for subsequent clinical research on the development of
monoclonal antibodies [145-148] and utilization of a monoclonal antibody cocktail such as
MB-003 [149], ZMAb [150], and ZMapp [151] in the treatment of the EBOV infection in
NHPs. It is interesting to note that all three monoclonal antibody cocktails include one
antibody that binds to or close to the glycan cap and that two of the three monoclonal
antibody cocktails include at least one antibody that binds the GP1/GP2 interface, indicating
that these two regions may be especially important in protection against EBOV [148]. The
treatment window of monoclonal antibody therapy can be extended by the co-administration
of adenovirus-vectored interferon therapy. In a guinea pig model, monoclonal antibodies
combined with adenovirus-vectored interferon given three days after infection resulted in
100% survival, a significant improvement over either treatment alone [152]. A subsequent
study showed that such a combination therapy is capable of saving 100% of EBOV-infected
NHPs when initiated after the presence of detectable viremia along with symptoms [153].
(2) Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide)
Favipiravir is a broad-spectrum inhibitor of viral RNA polymerase that is able to inhibit the
replication of many RNA viruses. It is registered in Japan for the treatment of influenza virus
infection [154,155]. Favipiravir is able to suppress the replication of the EBOV in cell
culture. Favipiravir, initiated at day 6 after EBOV infection, induced rapid virus clearance,
reduced the biochemical parameters of disease severity, and prevented a lethal outcome in
100% of mice lacking the Type I interferon receptor [156]. Oral favipiravir taken twice daily
for 14 days is able to give 100% protection against an aerosol EBOV infection in an immunedeficient mice model [157,158]. The survival benefit was suboptimal in NHPs. Only one of
the six animals tested survived. Studies using dosages that are two to five times higher and
have duration longer than shown in influenza studies are being conducted for the human
EBOV infection [5]. BCX4430, a synthetic adenosine analogue with a viral RNA polymerase
inhibitor function, is active against the EBOV and Marburg virus in rodent and cell culture.
BCX4430 completely protects NHPs from the Marburg virus infection when administered as
late as 48 hours after infection [159,160].
(3) Chloroquine
The antimalarial drug chloroquine is able to increase endosomal pH. An acidic endosomal
environment is important for the pH-dependent activation of cysteine proteases CatB and
CatL, the proteases responsible for the cleavage of EBOV GP1,2 essential for endosomal
virus-host membrane fusion [35,39,161-163]. However, proteolytic processing of the EBOV
glycoprotein has been demonstrated to be not critical for EBOV replication in cell culture
[164] or NHPs [165]. A recent study using a CatB and CatL deficient mouse model for the
study of the EBOV infection demonstrates that CatB and CatL activity is not absolutely
required for EBOV replication. The EBOV glycoprotein cleavage seems to be mediated
through a broader spectrum of proteases making therapeutic approaches targeting limited
proteases unlikely to be beneficial to combat EBOV infections [166]. A broad-spectrum
small molecule that targets the CatL cleavage of the EBOV and inhibits the entry of a wide
variety of viruses has recently been identified. It has been examined for the potential to
develop into a potent broad-spectrum antiviral medication [167].
(4) Cationic amphiphiles
Multiple cationic amphiphiles including amiodarone, dronedarone, verapamil, clomiphene,
and toremifene have been identified as potent inhibitors of the entry of the EBOV in an
NPC1-dependent fashion [38,168]. Amiodarone used for the treatment of atrial fibrillation
and ventricular cardiac arrhythmia can induce lipidosis with features similar to Niemann-Pick
C disease [169]. Amiodarone and dronedarone, having basic pKa and high water solubility at
acidic pH, accumulates within late endosomal compartments, blocking fluid-phase
endocytosis, proteolysis and lipid trafficking, and inducing a Niemann-Pick C-like
phenotype. In contrast to the Niemann-Pick type-C disease, they are not alleviated by
cholesterol removal [170,171].
Amiodarone, at concentrations that are routinely reached in human serum during antiarrhythmic therapy (1.5–2.5 µg/ml), is a potent inhibitor of filovirus cell entry through late
endosomes (IC50 0.25 µg/ml for EBOV), when induced as a Niemann-Pick C-like
phenotype. Significant inhibition is observed in most endothelial and epithelial cells (e.g.
macrophage, monocyte, vascular endothelial cell), except for primary hepatocyte and
fibroblast. The inhibitory effect of amiodarone on the entry of the EBOV was dose-dependent
and reversible upon removal of the drug. Prolonged exposure to amiodarone will not lead to a
compensatory change in the host cell. A similar inhibitory property is observed with the
amiodarone-related agent dronedarone and the L-type calcium channel blocker verapamil
[38,168,172,173].
Both clomiphene and toremifene have anti-EBOV activity in both the Vero E6 (interferondeficient African green monkey kidney epithelial cells) and HepG2 (human hepatocellular
carcinoma) cell lines. The anti-EBOV activity of clomiphene and toremifene is dependent not
on its estrogen receptor antagonistic action but upon the ability of both drugs to induce a
Niemann-Pick C-like phenotype to inhibit viral entry at late endosome. Clomiphene and
toremifene do not disrupt the interaction between primed GP1 and NPC1, but mediate the
entry block indirectly through NPC1 by targeting other endosomal/lysosomal proteins
involved in the cholesterol uptake pathway whose functions may be regulated by NPC1.
Clomiphene and toremifene at 60 mg/kg every other day have been shown to result in a 90%
and 50% survival rate, respectively, in EBOV-infected mice compared with 100% mortality
in the control group in an in vivo murine EBOV infection model. They are effective in both
male and female mice [38,174]. However, the therapeutic dose against EBOV cannot be
achieved with the oral clomiphene dose used for inducing ovulation in humans [175-177].
The therapeutic dose against EBOV with tolerable side effects can be achieved with
toremifene at an oral dose used in the human trial for the treatment of advanced carcinoma of
the breast [178-181]. Toremifene is well absorbed and >99.5% bound to plasma protein.
Toremifene undergoes extensive liver metabolism and enterohepatic recirculation. The
majority of the toremifene dose is excreted as metabolites in feces. The long terminal half-life
of oral toremifene may be due to both plasma protein binding and enterohepatic recirculation
[182,183].
(5) Interferon-beta
Interferon-induced transmembrane proteins (IFITMs) are expressed basally in the absence of
IFN induction in both primary tissues and cell lines [184]. An IFITM is able to inhibit the
entry of viruses to the host cell cytoplasm; permit endocytosis, but prevent subsequent viral
fusion; and release viral contents into the cytosol. The human IFITM locus is located on
chromosome 11 and composed of four functional genes: IFITM1, IFITM2, IFITM3, and
IFITM5. IFITM4p is a pseudogene. Viruses that are restricted by IFITM proteins tend to fuse
with host cell membranes in a late endosome or lysosome that precedes the induction of Type
I IFN in infected cells. Viral escape from restriction by IFITM proteins could be more
challenging than for antagonizing inhibitory factors that function at later stages of the virus
life cycle because the opportunity for de novo synthesis of viral inhibitors is not available. All
four human IFITM proteins are induced robustly by both Type I and Type II IFNs. IFITM1 is
active against multiple viruses, including the EBOV and hepatitis C viruses [185-187]. IFN-β
is able to induce interferon-inducible transmembrane protein production to restrict entry of
the EBOV [188]. Early post-exposure treatment with IFN-β significantly increased survival
time of rhesus macaques infected with a lethal dose of the EBOV, although IFN-β alone
failed to alter the mortality rate. IFN-β treatment was associated with a trend towards lower
plasma and tissue viral burden and pro-inflammatory cytokines production [56].
(6) Na+/K+ exchangers (amiloride and its derivatives)
Amiloride and its derivatives are used as potassium-sparing diuretics to treat hypertension
and congestive heart failure. Apart from inhibiting epithelial Na+ channel and cellular Na+/K+
exchangers, these drugs could also affect the function of other less well-defined ionexchangers (Na+/Ca2+ and Na+/Mg2+), and disturb the equilibrium of other ions, such as Mn2+
[189-192]. The entry of the EBOV into host cells is the first step of infection and a crucial
determinant of pathogenicity. Upon receptor binding between GP1 and host TIM-1 receptors,
the EBOV is internalized into endosomes primarily via the macropinocytic pathway.
Amiloride is able to inhibit the uptake of many viruses that utilize the macropinocytic
pathway for host cell entry [193-196]. Amiloride at non-cytotoxic dosages leads to potent
dose-dependent inhibition of the entry and infection of the EBOV [197,198]. Amiloride can
lead to dose-dependent inhibition of RNA synthesis. This may be due to a direct blockage of
a nucleotide entry tunnel or catalytic site, or due to its effect on the equilibrium of Mg2+ and
Mn2+ that are essential co-factors for polymerase activity and nucleotide insertion [199,200].
These novel antiviral mechanisms of amiloride may uncover new targets for drug discovery
against the EBOV.
(7) Na+/K+-ATPase pump inhibitors (ouabain, digoxin, and digitoxin)
Adenosine triphosphate (ATP) is essential in multiple steps in the replication cycle of many
viruses. Na+/K+-ATPase pump is located in the plasma membrane of all animal cells to
maintain the cell membrane potential. Budding of enveloped viruses is a complex
phenomenon that requires concerted actions of many viral and host components. ATP may
affect multiple steps in the budding process [201]. ATP is required for the assembly and
maturation of a number of enveloped viruses such as the influenza virus, vaccinia virus,
retrovirus, and herpes simplex virus. The Na+/K+-ATPase pump inhibitors, ouabain,
Lanatoside C, strophanthidin, and digoxin are able to inhibit the replication of the influenza
virus, Newcastle disease virus, and vesicular stomatitis virus through an interferonindependent mechanism [202]. Digoxin and Lanatoside C have been shown to inhibit
vaccinia virus replication at non-cytotoxic doses [203]. Ouabain has shown antiviral activity
against the influenza virus [204], herpes simplex virus [205], Sendai virus [206], murine
leukemic virus [207], cytomegalovirus porcine reproductive and respiratory syndrome virus
[208], and human cytomegalovirus virus [209]. One common feature shared by these viruses
is that they all possess a lipid envelope. The EBOV is an enveloped filamentous RNA virus.
The secondary matrix protein VP24—apart from its role in the evasion of host immune
response, nucleocapsid formation, and regulation of replication—has an important role in
viral budding and egress. Na+/K+-ATPase ATP1A1 is detected to have a close interaction
with VP24 of EBOV during replication. Ouabain, at a non-cytotoxic concentration of 20nM,
is able to suppress the replication of the EBOV in human MRC-5 cells [210,211]. Among the
three cardiac glycosides that may include digoxin, digitoxin, and ouabain, only digoxin is
commonly used in clinical practice. Ouabain, because of its poor oral availability, is used
primarily as a research tool. Further research should be conducted to investigate whether
digoxin and other Na+/K+-ATPase inhibitors might play a role in the management of the
EBOV or other enveloped virus infections.
(8) Antioxidants
The virus-associated glycoprotein GP1,2 is responsible for the activation of human
macrophages [13]. The highly glycosylated mucin-like region of the GP1 subunit of GP1,2 is
cytotoxic to the host cells [14]. The mucin-like region in GP1 leads to an accumulation of
GP1,2 at the endoplasmic reticulum, induces endoplasmic reticulum stress [212], and
activates nuclear factor kappa B (NF-κB) [213]. Mutations of the EBOV that lead to an
enhanced accumulation of GP1,2 in the endoplasmic reticulum were significantly more
cytotoxic than wild-type virus [214]. In human cells, the accumulation of protein in the
endoplasmic reticulum will lead to endoplasmic reticulum overload response (ER-overload)
which activates NF-κB through the production of ROS [215]. As a major transcription factor
for antiviral and immune stimulatory activities, NF-κB is thought to play an important role in
the induction of pro-inflammatory molecules, such as interleukin-1β (IL-1β), and tumor
necrosis factor α (TNF-α), upon cellular responses against a virus infection [216]. The
cytokine dysregulation of the EBOV involves massive ROS, NF-κB, TNF-α, and IL-1β
activation [65,66]. The effectiveness of antioxidant therapy for the EBOV infection indicates
the importance of ROS in the pathogenesis of the EBOV [217]. The activation of NF-κB by
ER-overload is ROS-dependent [218]. NF-κB-induced cytokine dysregulation of novel H1N1
pneumonia has been shown to be suppressible by high-dose N-acetylcysteine (NAC)
antioxidant therapy at 100 mg/kg continuous infusion daily [219]. Given the poor oral
availability of NAC in the range of 6% to 10% in humans [220], a therapeutic dose of NAC
equivalent to the intravenous route can hardly be delivered by oral preparation. NAC is a
category B drug for pregnancy and is affordable, with a wide therapeutic window. NAC has
an established safety profile even in high doses and prolonged use in humans [221-223].
Cytokine dysregulation is a common feature in the EBOV infection and is associated with an
enhanced mortality [65-68]. Antiviral medications directed against the mutable viral
determinants of the EBOV cannot directly prevent cytokine dysregulation. The early
endothelial vascular damage characteristic of the EBOV infection is not a direct effect of
virus-induced cytolysis of endothelial cells, but is due to cytokine dysregulation resulting
from massive release of proinflammatory cytokines/chemokines and ROS by infected
macrophage and monocytes [70-72]. Lymphocytes are resistant to the EBOV infection.
Cytokine dysregulation may also contribute to the diffuse bystander apoptosis of
lymphocytes [63,87-89]. With the safety profile of NAC, if the therapeutic efficacy of a highdose NAC antioxidant therapy to manage EBOV-induced cytokine dysregulation is
confirmed, it may revamp the future management of the EBOV infection.
Proposed prophylactic and therapeutic regimen against the Ebola virus
infection
There is a desperate need for a viable treatment regimen in Africa to engender hope and
encourage people with symptoms and their close contacts to seek medical treatment, so as to
limit the spread of the disease. This also helps to recruit and maintain adequate medical staff
who are at high risk of contracting the disease. A proposed regimen against the human EBOV
infection based on available medications and information from in vivo animal testing and in
vitro cell culture is attached (see Tables 2 and 3). This regimen contains a cocktail of
currently available medications that can target the different steps in the replication cycle of
the EBOV aiming to suppress viral proliferation. It has been shown that viral load is major
contribution to survival in both human and animal studies [60-62,136]. Through viral load
suppression, we may be able to prolong a patient’s survival in order to allow the development
of natural body immune defense against the EBOV.
Table 2 Proposed therapeutic regimen for the prophylaxes and treatment of human
EBOV infection based on available therapeutic medications and information from in
vivo animal testing and in vitro cell culture
Therapeutic regimen based on available medications for ebola virus prophylaxes and treatment
Ebola Virus
Available Medications
1
Prophylaxis
Amiodarone (macrophage, monocyte & endothelial cell)
Post Needle Stick Injury
IFN-β + amiodarone (macrophage, monocyte & endothelial cell) + toremifene (liver) 2,3 +
Prophylaxis
favipiravir4 ± convalescent blood serum
Treatment
Amiodarone (macrophage, monocyte & endothelial cell) + toremifene (liver) 2 ,3 + favipiravir4 +
high dose N-acetylcysteine infusion5 + convalescent blood serum + supportive care
1. 1 ml of blood may contain 10 9 to 10 10 virions in terminally ill patient. Prophylactic
amiodarone therapy may protect macrophage, monocyte and endothelial cells immediately
from EBOV during needle stick injury and accidental exposure and allow time for the
consideration of IFN-β, toremifene, favipiravir and convalescent blood serum therapy.
2. Amiodarone is unable to protect hepatocyte from EBOV infection.
3. Both amiodarone and toremifene can increase the risk of QT prolongation and Torsades de
pointes.
4. The recommended dosage for treatment of human EBOV infection may be 2 to 5 times
higher than influenza studies. Please confirm the recommended dose with the drug company.
5. N-acetylcysteine intravenous infusion at 100 mg/kg/day to control cytokine dysregulation
(e.g. add 5 g of intravenous preparation of N-acetylcysteine into each liter of intravenous
replacement fluid).
Table 3 Prophylaxis regimen for healthcare worker after needle stick injury
Regimen
Central Venous Line
Interferon-beta
Amiodarone
Toremifene
Oral1
Not Required
6 million international units (MIU) prefilled
pen via intramuscular injection (IMI) weekly
for 3 weeks.2
600 mg p.o. twice daily for 8 days (loading)
then maintenance 600 mg p.o. daily for
further 3 weeks.
Intravenous4
Required
6 MIU intravenous infusion over 2 hour daily for up to 3
weeks3 or 6 MIU prefilled pen IMI weekly for 3 weeks.
150 mg into 100 ml D5 over 10 minutes followed by 360 mg
infusion over 6 hours then 540 mg infusion over 18 hours
D1.4 Amiodarone 720 mg infusion daily or 600 mg p.o. twice
daily for further 7 days followed by 600 mg p.o. maintenance
daily for further 3 weeks.
800 mg p.o. on Day 1 (loading) then 400 mg p.o. daily.5
800 mg p.o. on Day 1 (loading) then 400 mg
p.o. daily.5
Favipiravir
1800 mg p.o. twice daily on Day 1 (loading
1800 mg p.o. twice daily on Day 1 (loading doses) then 800
doses) then 800 mg p.o. twice daily.6
mg p.o. twice daily.
1. Oral regimen are for those workers who are already on amiodarone prophylaxis with a loading dose of amiodarone 600
mg p.o. twice daily for 8 days followed by maintenance amiodarone 600 mg p.o. daily. Electrocardiogram and thyroid
function should be monitored.
2. Monitor for side effect of thrombocytopenia and proteinuria.
3. Intravenous dosage of IFN-β that are used for human hepatitis C virus infection to induce IFITM1 to limit viral entry.
4. Intravenous regimen is for those workers who have not been on amiodarone prophylaxis and agreed for the insertion of a
central venous line for drug administration. Intravenous amiodarone should be administered via central venous line to avoid
phlebitis. The dosage for treatment of frequently recurring ventricular fibrillation and hemodynamically unstable ventricular
tachycardia is recommended because it can achieve therapeutic drug level immediately after the first dose of amiodarone.
5. http://www.pulmcrit.org/2014/08/could-estrogen-receptor-antagonists.html.
6. Dosage for the treatment of human influenza virus infection in human Phase 3 trial of Favipiravir (FAVOR Study).
http://www.clinicaltrials.gov/show/NCT02008344.
The recommended dosage for treatment of human EBOV infection may be 2 to 5 times higher than influenza studies. Please
confirm the recommended dose with the drug company.
The EBOV has undergone a rapid mutation during its spread through humans [224-226]. The
EBOV is an RNA virus the replication of which is highly error prone with nearly one viral
mutation occurring during each cycle of replication. This extremely high mutation rate leads
to significant genetic and antigenic diversity that allows the EBOV population to evolve
resistance to antiviral medications and vaccines [227,228]. A combination therapy has been
used in the treatment of RNA virus infections, such as the human immunodeficiency virus
(HIV) [229,230] and hepatitis C [231,232] to minimize the development of drug resistance.
Given the broad cell tropism and high replication rate of the EBOV due to the potent
suppression of both innate and adaptive immune responses of the host, patients with the
EBOV infection have an extremely high viral load. The selective pressure in the presence of
the high mutation rate and viral load during the human EBOV infection make the evolution
of the EBOV viral strains resistant to a single drug inevitable. The currently available
medications in the proposed regimen—which is a treatment regimen containing a cocktail of
antiviral medications targeting the different steps of the EBOV replication in order to achieve
maximal suppression of viral replication and to prevent the rapid development of resistance
to favipiravir, the only drug in the regimen that is directed against a mutable target of the
EBOV—has been shown to reduce the replication of the EBOV. [233-235].
The current EBOV vaccine (rVSV-EBOGP and rChAd-EBOGP) and therapeutic agents
(ZMapp, TKM-Ebola, PMO AVI-6002, and favipiravir) under development are directed
against the mutable targets of the EBOV, and their effectiveness is limited by viral mutation.
The EBOV, being a RNA virus with limited coding capacity, has utilized the host’s unique
metabolic pathway for its viral entry, replication, and egress. Most of the therapeutic agents
in this current review are directed against non-mutable targets of the host which is
independent of viral mutation. These medications are FDA-approved for the treatment of
other diseases. They are available and stockpileable for immediate use. They may also have a
complementary role to those therapeutic agents under development that are directed against
the mutable targets of the EBOV.
The primary target of the EBOV is the mononuclear phagocytic system. The spectrum of
target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other
cells during the advanced stage of the disease [6,236,237]. The EBOV may produce a viral
load of up to 1010 virions per ml serum in terminally ill patients [80]. Oral amiodarone
prophylaxis, by inducing a Niemann-Pick C-like phenotype in the cells of the mononuclear
phagocytic system, may prevent viral entry into these cells during needle stick injury.
Through protection of the mononuclear system by our prophylaxis and cocktail therapy, we
hope to offer a better chance of survival to these patients by allowing them to develop a
natural body immune defense against the EBOV infection. The liver, containing the largest
number of fixed tissue macrophages (Kupffer cells), as part of the reticuloendothelial
immune defense system of the body, is a major target for the EBOV infection [238,239]. The
EBOV replicates to high titer in the liver [240]. Hepatic apoptosis may play a role in the
pathogenesis of the EBOV infection [88]. Toremifene is added to the treatment regimen for
hepatic protection because amiodarone does not exert inhibitory action against the EBOV in
hepatocyte. However, both amiodarone and toremifene can increase QTc and the risk of
Torsades de pointes. Therefore electrocardiogram should be carefully monitored if both drugs
are to be used. Amiodarone, favipiravir, and toremifene are available and stockpileable in
oral preparations. These properties are advantageous in outbreak situations and contingency
planning of a potential EBOV epidemic or pandemic. The avoidance of intravenous
administration will prevent needle stick injury in healthcare workers caring for the infected
patients.
IFN-β may have potential as an adjunctive post-exposure therapy for high-risk exposure, such
as needle stick injury, by inducing IFITM1 to limit entry of the EBOV. Post-exposure IFN-β
treatment was associated with a trend towards lower plasma and tissue viral burden and proinflammatory cytokines production [56]. The reduction in viral load and cytokine
dysregulation coupled with optimal supportive therapy may improve the chance of survival of
the host to allow the development of natural immunity to control the underlying EBOV
infection. IFITM1 is active against multiple viruses, including the EBOV [185,188] and
hepatitis C [186,187,241,242]. Interferon induced IFITM1 plays an important role in the
treatment of human HCV infection by inhibiting entry of HCV into the host cell [243]. Six
million international units (MIU) of IFN-β intravenous administration is as effective as a
three MIU twice-daily regimen for treatment of the HCV infection [244], but has lesser side
effects that require discontinuation of the medication [245,246]. As the aim of IFN-β therapy
in our regimen for post needle stick prophylaxis against the EBOV infection is to induce
IFITM1 to limit viral entry, the dose of IFN-β for the post needle stick prophylaxis [247,248]
or induction therapy [249,250] for HCV infection in humans is chosen. Once infection is
fully established, IFN-β are replaced by convalescent blood serum and high-dose NAC
infusion for providing passive humoral immunity and for the control of ROS-dependent NFκB-induced cytokine dysregulation respectively.
Summary
The EBOV is classified as biosafety level 4 pathogen and is classified by Centers for Disease
Control and Prevention as a category A agent of bioterrorism with no approved therapies and
vaccines for its treatment but carrying a high potential for large-scale dissemination. Recent
political, economic, military, and religious turbulence around the world raises concerns that
the EBOV might be used as an agent of bioterrorism [251-253]. The recent EBOV epidemic
is spiraling out of control in West Africa. The containment measures that worked in the past,
such as isolating those who are infected and tracing their contacts, have failed due to an
exponential rise in infected patients. Although the short-term (three- and six-week)
probability of international spread outside the African region is small, the risk of the
extension of the outbreak to other African countries followed by international dissemination
on a longer time scale is not negligible, indicating that this public health emergency has the
potential to grow to extraordinarily destructive dimensions [254,255]. Although several
promising therapeutic agents and vaccines against the EBOV are undergoing the Phase I
human trial, the current epidemic might be outpacing the speed at which drugs and vaccines
can be produced [5]. To combat such an unprecedented global public-health crisis before
these experimental agents are available, alternative available interventions capable of
managing the enhanced viral replication and cytokine dysregulation of the human EBOV
infection should be explored and stockpiled as contingency preparation for the worst-case
scenario of an impending human EBOV pandemic [256].
Like all viruses, the EBOV largely relies on host cell factors and physiological processes for
its entry, replication, and egress which, in turn, lead to cytopathic damage, cytokine
dysregulation, and death of the host. These non-mutable key steps inside the host may be
novel targets for future therapeutic strategies against these rapidly mutating viruses. If the
efficacy of amiloride, digoxin, amiodarone, and high-dose NAC antioxidant therapy against
the human EBOV infection is confirmed, the availability and affordability of these
stockpileable agents make them ideal medications in pandemic situation and in countries with
limited resources. They may have a complementary role to other antiviral medications to
prevent the emergence of resistant strains. This may also signify a major breakthrough in
future management of the EBOV infection.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KYL and GWYN contributed to the conception, drafting, and writing of the paper. KYL,
GWYN and FC revised the draft paper. All authors read and approved the revised paper.
Acknowledgement
This paper is dedicated to Dr Lillian Lai Lan Fong (15/9/1943 – 22/7/2014), the founder of
the Intensive Care Unit of Queen Elizabeth Hospital, Hong Kong.
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Additional file
Additional_file_1 as PDF
Additional file 1 Multilingual abstracts in the six official working languages of the United
Nations.
Figure 1
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